INTRODUCTION:

Bacteriophages are the world’s most abundant organisms. It’s estimated that the total number of bacteriophages on Earth exceeds ten thousand billion billion. They prey only on bacteria, never on human cells or those of any more complex organism. Even among their bacterial hosts, bacteriophages are highly specific, with most infecting only a single species of bacteria. In many cases, only specific strains within that species are infected.

The human oral cavity provides the perfect portal of entry for viruses and bacteria in the environment to access new hosts. Hence, the oral cavity is one of the most densely populated habitats of the human body containing some 6 cells billion bacteria and potentially 35 times that many viruses. The role of these viral communities remains unclear; however, many are bacteriophage that may have active roles in shaping the ecology of oral bacterial communities. Other implications for the presence of such vast oral phage communities include accelerating the molecular diversity of their bacterial hosts as both host and phage mutate to gain evolutionary advantages.

Viruses that attack bacteria were observed by Twort and d'Herelle in 1915 and 1917. They observed that broth cultures of certain intestinal bacteria could be dissolved by addition of a bacteria-free filtrate obtained from sewage. The lysis of the bacterial cells was said to be brought about by a virus which meant a "filterable poison" ("virus" is Latin for "poison").

Probably every known bacterium is subject to infection by one or more viruses or "bacteriophages" as they are known ("phage" for short, from Gr. "phagein" meaning "to eat" or "to nibble"). Most research has been done on the phages that attack E. coli, especially the T-phages and phage lambda.

Like most viruses, bacteriophages typically carry only the genetic information needed for replication of their nucleic acid and synthesis of their protein coats. When phages infect their host cell, the order of business is to replicate their nucleic acid and to produce the protective protein coat. But they cannot do this alone. They require precursors, energy generation and ribosomes supplied by their bacterial host cell.

Phages are widely distributed in locations populated by bacterial hosts, such as soil or the intestines of animals. One of the densest natural sources for phages and other viruses is sea water, where up to 9×108 virions per milliliter have been found in microbial mats at the surface, and up to 70% of marine bacteria may be infected by phages. They have been gused for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria. Nevertheless, phages of Inoviridae have been shown to complicate biofilms involved in pneumonia and cystic fibrosis, shelter the bacteria from drugs meant to eradicate disease and promote persistent infection.

There are two major types of bacteriophages. Virulent phages destroy their host as a result of reproduction. Examples of these include the T-phages, or T1-T7. These phages commonly infect E. coli bacteria. Temperate phages brainwash their host and take over its machinery but don't actually kill it. Examples of these phages are lambda types.

Once the bacteriophage has infected the host cell, the resulting infection may be either lytic, reprogramming and then destroying the infected cell, or lysogenic, where the bacteriophage genome is integrated into the bacterial genome and passed on to future generations of bacteria. Lysogenic bacteriophages may also reactivate, producing a new generation of viruses.

Persistence in human body

Early studies of bacteriophage in the oral cavity identified phage that parasitise oral pathogens such as Aggregatibacter actinomycetemcomitans . In these studies, the presence of A. actinomycetemcomitans phage was positively correlated with rapidly destructive periodontitis , which suggested a role for oral phage in bacterial virulence. However, other studies showed that these phage were not associated with periodontal disease , so their role in the oral micro-biome is still unclear. Regardless of their role in oral disease, previous studies show that phage in the oral cavity can act both as commensals and pathogens which suggests that they play significant roles in the ecology of the human oral.

Studies also have identified some eukaryotic viruses including torque teno viruses, circoviruses, herpesviruses (HSV), and Epstein–Barr virus (EBV) among a few others, but phage appear to be more highly abundant, which may reflect the high ratio of bacterial cells to our own cells in the oral cavity. [1]

Early studies

Early studies of phage in the human oral cavity relied upon the presence of virus-like pa Letarov A., Kulikov E. (2009). The bacteriophages in human- and animal body-associated microbial communities. J. Appl. Microbiol. 107, 1–13 articles (VLPs) using electron microscopy to speculate that there may be many phage present in dental plaque. Because these types of studies could not also taxonomically characterise the phage present, it was unclear whether the presence of VLPs represented a few relatively abundant phage or many different evenly distributed phage. Using epifluorescence microscopy, studies have shown that there are approximately 108 VLPs per mL of fluid from oropharyngeal swabs , 108 VLPs per mL of saliva , and 107 VLPs per milligram of dental plaque . Culture- and morphology-based techniques have not been sufficient to characterise the diversity of phage in the oral cavity, but the utilization of metagenomics techniques based on shotgun sequencing approaches have proven effective in uncovering the membership and diversity of oral phage communities .

By using next generation sequencing approaches, such as metagenomics, we now recognise that the oral cavity is home to a large population of viruses, many of which can be identified as bacteriophage. These studies also have identified some eukaryotic viruses including torque teno viruses, circoviruses, herpesviruses (HSV), and Epstein–Barr virus (EBV) among a few others, but phage appear to be more highly abundant, which may reflect the high ratio of bacterial cells to our own cells in the oral cavity. Another potential explanation for the abundance of phage compared to eukaryotic viruses are enrichment techniques such as cesium chloride (CsCl) density gradient centrifugation and sequential filtration, which could result in technical biases by removing viruses from the oral virome. Enveloped viruses such as HSV and EBV have been found in the oral virome, indicating that enveloped viruses may be identified, but larger viruses such as mimiviruses may be trapped by filtration. Smaller viruses such as human papillomaviruses are readily detected after CsCl gradient enrichment so the extent of virion size biases is difficult to quantify for smaller viruses. Most studies of human viromes have focused only on DNA viruses, so the constituents and potential roles of RNA phage communities lags significantly behind . Many phage in the oral virome may be intracellular at the time of analysis, and thus, they could also go unrecognised or their relative abundance underestimated in oral virome analysis.

Diversity

While technical biases such as the concentration on DNA viruses and utilization of filtration techniques could lead to substantial underestimations of oral phage community diversity, there are other factors that might contribute to the overestimation of oral phage diversity. Principally among these factors include under sampling of the phage community and an inability to properly assemble phage genomes from complex communities.. One means of estimating the diversity of viral communities is a tool called Phage Communities from Contig Spectrum (PHACCS),, which uses a rank-abundance model based on the full spectra of assembled and size-sorted contigs. Use of this tool has recently highlighted the critical role that assemblers play in estimating phage community diversity, by demonstrating that certain assemblers may provide significantly different estimations of community diversity. Despite the limitations imposed through the assembly process, estimates of phage diversity in human saliva suggest that there are hundreds to thousands of different phage genotypes that are relatively evenly distributed in this particular environment . These results indicate that the numerous VLPs in the oral cavity likely represent many different evenly distributed phage. Another study showed that phage community diversity still was substantially overestimated in oral viromes likely as a result or limitations in the phage assembly process . An alternative method termed the Homologous Viral Diversity Index (HVDI) uses homology amongst assembled phage contigs to identify contigs through network analysis that likely belong to the same viruses . A corrected contig spectra is formed from the networks and utilised to reduce the overestimation of phage genotypes. Utilization of this method indicates that phage are less evenly distributed in the human oral cavity than were originally projected . It also shows that phage diversity in the oral cavity is relatively homogenous between different human subjects and is far greater than is estimated in the colon.

There are numerous different mechanisms by which oral bacteriophage may evade their host immune systems and persist. These include but are not limited to carrying their own restriction/modification enzymes and avoiding cognate sequences for restriction/modification systems. [2]

Vaccines

Trillions of bacteriophages have been administered to millions of people as part of live organism vaccines around the globe.. In 1975, the realisation that numerous human vaccines were contaminated with bacteriophage resulted in a high-profile exemption being issued by the US Food and Drug Administration (FDA). This effectively meant that FDA was to formally approve the inclusion of bacteriophages of various and un- known kinds in human vaccines. [3]

FDA statement

Some years later, this resulted in FDA’s tolerance of vaccine contaminants in a court case in 1987, that stated:

a) “Each seed virus used in manufacture shall be demonstrated to be free of extraneous microbial agents except for unavoidable bacteriophage.”

b) In 1973, scientists at the Bureau of Biologics of the Food and Drug Administration (FDA) reported that all live virus vaccines are grossly contaminated with phage. This finding presented a problem since federal regulations forbade extraneous material in vaccines and no one knew whether phage are harmful to human beings or whether they could be removed from vaccines. The temporary solution was to amend the regulations so as to permit phage in vaccines.

As a direct consequence of these findings, namely, that bacteriophages were heavily contaminating human vaccines, the US FDA set about proving that bacteriophages as isolated from therapeutic vaccines were unlikely to cause harm to humans.[4]

Phage therapy

Phage therapy or viral phage therapy is the therapeutic use of bacteriophages to treat pathogenic bacterial infections. Phage therapy has many potential applications in human medicine as well as dentistry, veterinary science, and agriculture. If the target host of a phage therapy treatment is not an animal, the term "biocontrol" is usually employed, rather than "phage therapy".[5]

They would have a high therapeutic index, that is, phage therapy would be expected to give rise to few side effects. Because phages replicate in vivo, a smaller effective dose can be used. On the other hand, this specificity is also a disadvantage; a phage will only kill a bacterium if it is a match to the specific strain. Consequently, phage mixtures are often applied to improve the chances of success, or samples can be taken and an appropriate phage identified and grown.

Bacteriophages are much more specific than antibiotics. They are typically harmless not only to the host organism, but also to other normal flora, such as those in the gut, to reduce the opportunistic infection. Phages tend to be more successful than antibiotics where there is a biofilm covered by a polysaccharide layer, which antibiotics typically cannot penetrate. In the West, no therapies are currently authorised for use on humans, although phages for killing food poisoning bacteria (Listeria) are now in use.

Phages are currently being used therapeutically to treat bacterial infections that do not respond to conventional antibiotics, particularly in Russia and Georgia.] There is also a phage therapy unit in Wroclaw, Poland, established 2005, the only such centre in a European Union country.[6]

D'Herelle's commercial laboratory in Paris produced at least five phage preparations against various bacterial infections. Therapeutic phages were also produced in the United States. In the 1940s, the Eli Lilly. Company (Indianapolis, Ind.) produced seven phage products for human use, These preparations consisted of phage-lysed, bacteriologically sterile broth cultures of the targeted bacteria or the same preparations in a water-soluble jelly base. They were used to treat various infections, including abscesses, suppurating wounds, vaginitis, acute and chronic infections of the upper respiratory tract, and mastoid infections. Despite the large number of publications on phage therapy, there are very few reports in which the pharmacokinetics of therapeutic phage preparations is delineated. The few publications available on the subject suggest that phages get into the bloodstream of laboratory animals (after a single oral dose) within 2 to 4 h and that they are found in the internal organs (liver, spleen, kidney, etc.) in approximately 10 h. Also, data concerning the persistence of administered phages indicate that phages can remain in the human body for relatively prolonged periods of time, i.e., up to several days.[7]

Advantages

They are extremely specific to their target bacteria and won t affect other cells. This is especially useful if patients are allergic to antibiotics because phages will not affect any other cells than the ones targeted for infection .

Phages are known to outnumber bacteria 10:1, they can overpower bacteria and stop them from dividing and continuing to infect the host.

Phages mutate at a higher rate than bacteria and are able to respond fast to possible phage- resistant bacteria.

The cost of developing a phage system is cheaper than that of developing a new antibiotic.

Phages or their products do not have any side effects.

Phage therapy in dentistry

Bacteriophages lytic for a range of oral bacteria may be considered as a potential resource of bacteriophage therapy of oral infection. A study by Paisano et al vitro antimicrobial effect of bacteriophages on human dentin infected with Enterococcus faecalis showed that addition of phage lysate to the roots following the 6 day incubation period led to a substantial reduction in bacteria viability. They concluded that phage therapy may be an important alternative for the treatment of root canal infections refractory to conventional endodontic therapy. Keivan et al, have identified the Streptococcus salivarius bacteriophage isolated from Persian Gulf as a potential agent for dental caries phage therapy. They have isolated a lytic bacteriophage from Persian Gulf that attached specifically to Streptococcus salivarius, a member of dental caries producing Streptococci. The identification of new lytic phages capable to eliminate oral streptococci, starters of dental plaque formation, could be considered as a powerful approach for phage therapy of oral pathogenic bacteria. Recently the efficacy of bacteriophage treatment on Pseudomonas aeruginosa biofilms in a root canal model has been studied. It was seen that phage application significantly reduced the biomass of 24 hour and 96 hour PA4 biofilms grown on micro plates but did not produce significant reduction of 24 hour or 96 hour PA 14 biofilms grown in the extracted tooth mode.[8]

Partners in slime

Mucus used to be regarded as merely a physical barrier to prevent invading organisms from entering your body, as well as functioning as a lubricant. But recent scientific findings suggest it plays a far more active and critical role.

For some time, researchers have known that mucus is loaded with viruses.

But this new study found many of these viruses to actually serve as immune helpers—not enemies—and an important part of your body’s defence system. The study’s findings appear in the May 20, 2013 issue of Proceedings of the National Academy of Sciences.

Scientists bacteriophage are present in great numbers in virtually all mucus samples.

Wherever bacteria resided, phages were present , because phages depend on bacteria for their survival. In one of the study, microbiologist Jeremy Barr and colleagues noticed there were MANY more phages in mucus than in mucus-free zones, just millimetres away.

For example, in the saliva surrounding human gums, they found about five phages to everyone bacterium. But on the mucosal surface of the gum itself, the ratio was closer to 40 to one.

Phages specialise in breaking open and killing certain kinds of bacteria, hijacking them in order to replicate. Most phages have hollow heads, which store their DNA and RNA, and tunnel tails designed for binding to the surface of their bacterial targets. According to phages.org, once a phage has attached itself to a bacterium:

“The viral DNA is then injected through the tail into the host cell, where it directs the production of progeny phages, often over a hundred in half an hour. These 'young' phages burst from the host cell (killing it) and infect more bacteria.”[9]

REFERENCES:

1. Sustainability of virulence in a phage-bacterial ecosystem, Heilmann S, et.al, J Virol, 2010; 84: 3016–22.

2. New insights into the possible role of bacteriophages in host defence and disease, , Kinga Switala-Jeleń, Maria, Nowaczyk,Bio med central, the open access publisher, 2014, 8062–8070.

3. Pros and cons of phage therapy, Catherine Loc-Carrillo and Stephen T Abedon, Pubmed Mar-Apr; 1(2)

4. Bacteriophage therapy: an alternative to antibiotics, April Cashin-Garbutt, MA (Cantab), 322–329, 2009

5. Sphingolipids from a symbiotic microbe regulate homeostasis of host intestinal natural killer T cells, Olszal T, et al. (2014).

6. Marine viromes of four oceanic regions, Angly F. E., Felts B., Breitbart M., Salamon P., Edwards R. A., Carlson C., et al. (2006), The PLoS Biol. 368 10.1371

7. Prophage contribution to bacterial population dynamics , Bossi L., Fuentes J. A., Mora G., Figueroa-Bossi N, J. Bacteriol. 2003, 185, 6467–6471

8. Inducible prophages contribute to Salmonella virulence in mice, Figueroa-Bossi,. 33, 167–176.

9. The bacteriophages in human- and animal body-associated microbial communities, Letarov A., Kulikov J. Appl. Microbiol. 2009, 107, 1–13

Received on 16.06.2016 Modified on 24.06.2016

Research J. Pharm. and Tech 2017; 10(4):1204-1208.

DOI: 10.5958/0974-360X.2017.00216.5